Abstract

Cells have evolved biomolecular networks that process and respond to changing chemical environments. Understanding how complex protein interactions give rise to emergent network properties requires time-resolved analysis of cellular response under a large number of genetic perturbations and chemical environments. To date, the lack of technologies for scalable cell analysis under well-controlled and time-varying conditions has made such global studies either impossible or impractical. To address this need, we have developed a high-throughput microfluidic imaging platform for single-cell studies of network response under hundreds of combined genetic perturbations and time-varying stimulant sequences. Our platform combines programmable on-chip mixing and perfusion with high-throughput image acquisition and processing to perform 256 simultaneous time-lapse live-cell imaging experiments. Nonadherent cells are captured in an array of 2,048 microfluidic cell traps to allow for the imaging of eight different genotypes over 12 h and in response to 32 unique sequences of stimulation, generating a total of 49,000 images per run. Using 12 devices, we carried out >3,000 live-cell imaging experiments to investigate the mating pheromone response in Saccharomyces cerevisiae under combined genetic perturbations and changing environmental conditions. Comprehensive analysis of 11 deletion mutants reveals both distinct thresholds for morphological switching and new dynamic phenotypes that are not observed in static conditions. For example, kss1Delta, fus3Delta, msg5Delta, and ptp2Delta mutants exhibit distinctive stimulus-frequency-dependent signaling phenotypes, implicating their role in filtering and network memory. The combination of parallel microfluidic control with high-throughput imaging provides a powerful tool for systems-level studies of single-cell decision making.

Mating response to persistent α-factor stimulation. (A and B) WT time course data showing mean and variation of response to constant stimulation with 20 nM α-factor. Stimulated with α-factor at t = 0 is indicated by shading. (A) Measured GFP concentration, reporting mating-specific gene expression, of each cell, with mean of population indicated in red. (B) Number of cells in the chamber as a function of time showing arrest under α-factor stimulation. (C) Time course and dose response of mean GFP concentration in WT cells for all α-factor concentrations. (D) Strain comparison of signaling under constant stimulation. Initial dGFP/dt for all strains at the given concentrations relative to WT is shown (see SI Text). Initial dGFP/dt is calculated as the slope of a line fitted to the population averaged GFP concentrations between 30 and 180 min. (E) Performance of on-chip chemical formulation. Fluorescent measurements of 32 concentrations generated on-chip as detailed in SI Text and Table S1 are shown.

Morphological and transient stimulation responses. (A–C) Morphological response of WT (A), msg5Δ (B), ste50Δ (C) across all α-factor concentrations under constant stimulation. Color code for morphology shows representative images of yeast form cells (YF) at 3.2 nM (red); hyperelongated cells (HE) at 17 nM (blue), and shmoo cells (S) at 90 nM (green). The figures presents the mean GFP concentration, reporting mating-specific gene expression as a function of α-factor concentration for each classification, with dot opacity indicating the fraction of cells with that morphology. Error bars represent SD of measured GFP expression for cells of each morphology. Measurements were taken 360 min after exposure to pheromone. (D and E) WT time course response to a 180-min duration 50 nM α-factor pulse. Cells are stimulated with α-factor at t = 0; shading indicates the presence of α-factor. (D) GFP concentration, reporting mating-specific gene expression per cell with mean of population indicated in red with nearest-neighbor time point averaging used to smooth the mean curve. (E) Total number of cells vs. time showing transient growth arrest during α-factor pulse. Each blue diamond is the total number of cells in the microchamber array at a given time. (F) Representative data of population mean GFP concentration in response to transient α-factor pulse of varying duration. Data are shown for 20 nM α-factor condition.